Mg²⁺ and Ca²⁺ differentially regulate DNA binding and dimerization of DREAM

DREAM (calsenilin/KChlP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca²⁺ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca²⁺ binding to the various mutants revealed that, in the absence of Mg²⁺, Ca²⁺ binds independently and se-quentially to EF-3 (ΔH = -2.4 kcal/mol), EF-4 (ΔH = +5.2 kcal/mol), and EF-2 (ΔH = +1 kcal/mol). By contrast, only two Ca²⁺ bind to DREAM in the presence of physiologi-cal levels of Mg²⁺ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg²⁺. ITC measurements demonstrate that one Mg²⁺ binds enthalpically with high affinity (K_d = 13 μM and ΔH = -0.79 kcal/mol) and two or more Mg²⁺ bind entropically in the millimolar range. Size-exclusion chromatograpliy studies revealed that Mg²⁺ stabilizes DREAM as a monomer, whereas Ca²⁺ induces protein dinierization. Electrophoretic mobility shift assays indicated that Mg²⁺ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca²⁺ levels, suggesting that binding of a single Ca ²⁺ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca²⁺ and Mg²⁺ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg²⁺ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca ²⁺-induced protein dimerization disrupts DNA binding.